Biofabrication of ZnO/Malachite nanocomposite and its coating with chitosan to heal infectious wounds

Recently, nanocomposites produced from clays and metals coated with chitosan have shown wound healing activity. This study aimed to synthesize the zinc oxide/malachite nanocomposite (ZnO/Mlt-NC) and its coating form with chitosan (ZnO/Mlt/Chsn-NC). Physicochemical characterization of the produced nanocomposites was investigated. Biomedical effects of nanocomposites, such as in vivo and in vitro antibacterial activity, antioxidant properties, cytotoxicity, and modulation in the gene expressions of interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), and transforming growth factor-β (TGF-β) and histopathological parameters, were also investigated. Expression intensities of basic fibroblast growth factor (bFGF) and tumor necrosis factor alpha (TNF-α) were also investigated by immunofluorescence staining. To investigate biomedical effects under in vivo conditions, infected wounds were induced and inoculated with Staphylococcus aureus (ATCC 25923), and Pseudomonas aeruginosa (ATCC 27853). The results indicated spherical ZnO nanoparticles on the surface of malachite and strong antibacterial activity and antioxidant properties. The ointments produced from the nanocomposites also exhibited wound healing activity. The administration of the ointments prepared from ZnO/Mlt, and ZnO/Mlt/Chsn NCs decreased the expressions of IL-1β, IL-6, and TNF-α, while it increased the expressions of IL-10, TGF-β and bFGF. In sum, the nanocomposites produced from ZnO, malachite, and chitosan had better biological activity than ZnO/Malachite nanocomposites. We suggest applying ZnO/Mlt/Chsn nanocomposites in the structure of ointments to treat infected wounds after future clinical studies.


Abbreviations
Skin wounds cause damage to healthcare systems and loss economic 1 . Wounds are classified as acute and chronic based on the pathogenesis and consequences 2 . Acute wounds induce molecular processes to obtain structural integrity. Immune cells and factors play pivotal roles in acute wound healing 3 . The faulted regulation of the immune response results in the formation of chronic wounds 4 . Infectious wounds are a form of acute wounds characterized by the presence of bacteria in viable tissue and damage to tissues 5 . The infections start with bacteria colonization and can cause systemic infection. Staphylococcus aureus and Pseudomonas aeruginosa are the most common bacteria in infected wounds 6 . In infected wounds, the wound healing process is delayed 7 . Infected wounds also cause overproduction of reactive oxygen species and induce faults in antioxidant systems 8 . Antibiotics are used to treat infected wounds; however, they cause antimicrobial resistance. Therefore, it is essential to find safe and novel agents for the treatment of infected wounds 9 . Metal oxides nanomaterials are safe and cheap structures used to treat wounds 10 . Copper oxide is extensively used for wound healing activity owing to its biomedical properties like antibacterial activity 3 . Malachite is a form of copper oxide ore derivatives as Cu 2 CO(OH) 2 11 . It has been reported that malachite exhibits antibacterial activity against S. aureus and P. aeruginosa by changing the integrity of the cell membrane and antioxidant properties 12 . Recently, studies have reported synergistic interaction effects between clays and metal nanoparticles [13][14][15] . Metallic nanoparticles, including ZnO nanoparticles (ZnONPs), are extensively used in medicine. ZnONPs exhibit antioxidant properties via the electron donation property of the oxygen atom in ZnO nanomaterial and antibacterial properties via the electrostatic activity between the negative charge of bacterial cells and the positive charge of ZnO nanoparticles that can be used in the wound healing process 16 . ZnONPs also influence oxidative stress and increase the generation of reactive oxygen species 16 . They also promote the regeneration of damaged tissues by activating collagen synthesis 17 . Metal oxide NPs, including ZnO NPs, may be stabilized by being mixed with other inorganic structures like malachite 18,19 . This study aims to synthesize ZnO/Mlt-NC as an agent for the wound healing process using the external biofabrication method. This method requires plant active compounds, including phenolic compounds for capping and stabilizing [13][14][15] . In this study, we used pennyroyal (Mentha pulegium) extract to synthesize ZnO/Mlt-NC as a capping and stabilizing agent. M. pulegium belongs to the mint family, Lamiaceae. It contains some phenolic compounds, such as syringic acid, ferulic acid, and flavonoid compounds, including isorhamnetin-3-O-glucoside and kaempferol-3-O-rutinoside that can contribute to capping and stabilizing. Therefore, in the present study, for the first time, M. pulegium extract was used to synthesize ZnO/Mlt-NC 20 .
To improve biological properties, the synthesized nanocomposites were coated with chitosan. Chitosan is known to have antibacterial 21 and anti-inflammatory 22 properties and exhibits skin regenerative behavior, biocompatibility, and biodegradability 14,15,23 . It is also a safe compound and has very low toxicity 21 . It shows antibacterial activity via penetration in the cellular membrane 21 and clears free radicals via adsorption, ion-exchange, and chelation 13 .

Results and discussion
The results of the XRD pattern in Fig. 1E exhibited multiple obvious peaks for the malachite-based composite, which are similar to the peaks reported in previous studies 11 . In addition to malachite, a small amount of quartz was also observed. The results indicated strong and sharp diffraction lines originated from ZnO with a hexagonal    24 . Figure 1F shows the peak at 1644 cm −1 that is attributed to residual N-acetyl groups (C=O stretching of amide I) 13 . A band at 1558 cm −1 is observed for the N-H bending of the primary amine. The CH 2 bending and CH 3 symmetrical deformations were approved by bands found at around 1423 and 1393 cm −1 , respectively. Figure 1H presents a mixture of characteristic peaks due to presence of ZnO/Mlt-NC and Chsn. Figure 3A,B and D,E depict the FESEM images of malachite and as-synthesized ZnO/Mlt-NC, respectively. According to Fig. 3D, the thickness of the malachite plane is 79-116 nm. Furthermore, it can be observed that ZnO nanoparticles have a globular shape. Based on the FESEM image, the size of as-synthesized ZnO particles on the malachite plate ranges from 21 to 29 nm. According to the TEM images in Fig. 3C,F, the black spherical globules of ZnO are immobilized on the malachite.  Antibacterial results. Table 1 presents the results of MIC and MBC. The lowest bacteriostatic and bactericide activities are observed in the Mlt group for both bacteria. ZnO/Mlt-NC exhibited better bacteriostatic and bactericide activities than the Mlt group did for both bacteria. The highest bacteriostatic and bactericide activities were observed for ZnO/Mlt/Chsn-NC for both bacteria. All the nanocomposites were efficient on P. aeruginosa and S. aureus. ZnO/Mlt/Chsn-NC showed better antibacterial activity than the antibiotics did in MIC and MBC tests.

Investigation on colloidal properties of ZnO/Mlt-NC.
The results of the well test in Fig. 6 indicated that Mlt did not cause any inhibition zone for P. aeruginosa, but it formed a small inhibition zone for S. aureus (0.50 ± 0.707 mm). ZnO/Mlt nanocomposite had a higher inhibition zone than Mlt. The results revealed that ZnO/Mlt created an inhibition zone of 21.50 ± 1.41 mm for S. aureus, while it formed an inhibition zone of 18.50 ± 1.41 mm for P. aeruginosa (P = 0.021). The highest inhibition zone was observed in ZnO/Mlt/Chsn-NC than in the other nanocomposites. The results demonstrated that ZnO/Mlt/Chsn-NC created inhibition zones of 25.25 ± 2.12 mm and 23.25 ± 1.41 mm for S. aureus, and P. aeruginosa bacteria, respectively, which were significantly different(P = 0.0285). The diameters of the bacitracin antibiotic zone as a standard for S. aureus and the polymixin B antibiotic as a standard for P. aeruginosa were 20.10 ± 1.83 mm and 18.21 ± 1.12 mm for S. aureus and P. aeruginosa bacteria, respectively, which were significantly different (P = 0.0375). The results did not exhibit significant differences (P > 0.05) between ZnO/Mlt/ Chsn-NC and antibiotics for the zone diameter.
The results of the kinetic section demonstrated that all nanocomposites showed antibacterial activity from 1 to 24 h. Mlt, ZnO/Mlt, and ZnO/Mlt/Chsn nanocomposites demonstrated antibacterial activity at all times. The results of the kinetic time confirmed other antibacterial results. Based on kinetic findings, ZnO/Mlt/Chsn-NC exhibited the most powerful antibacterial activity, so that ZnO/Mlt/Chsn-NC decreased bacteria colony from 3 h after incubation, and the colony number was almost zero in 12 h after incubation; antibiotics showed similar effects after 24 h, and ZnO/Mlt and antibiotics exhibited similar effects.
As the results show, NCs exhibited antibacterial activity via the release of LDH and production of ROS (Fig. 7). The results showed that exposing bacteria to NCs increased the release of LDH compared to the control treatment (P = 0.0001). Production of ROS was greater in NCs than in the control treatment (P = 0.0001). Parallel with other findings, the greatest release of LDH was observed in bacteria exposed to ZnO/Mlt/Chsn, ZnO/ Mlt, Mlt and control groups, respectively. Production of ROS was also greater in ZnO/Mlt/Chsn, ZnO/Mlt, Mlt and control groups.   www.nature.com/scientificreports/ known to have antibacterial properties by mechanisms, including penetration in the cellular membrane 13,15,21 .
In the current study, the release of LDH and the production of ROS were investigated. Based on findings, NCs penetrate into bacterial membrane integrity, disrupt it, and increase the release of LDH in bacteria. In addition, the produced ROS directly attacks bacterial membrane, destroys it, and finally causes cellular death. The results show that Mlt, ZnO, and Chsn have a synergism interaction effect on antibacterial activity. The effects of ZnO/ Mlt/Chsn-NC were almost similar for both bacteria, revealing its efficiency for both bacteria.
DPPH results. Figure 27 showed that malachite at higher concentrations exhibited cytotoxicity owing to the involvement in antioxidant activity. The results showed that addition of ZnO decreased cytotoxicity that could be attributed to coating effects of ZnO. Gharehpapagh et al. 13 showed that nanoparticles had lower cytotoxicity that coated the surface of the malachite and decreased its toxicity. Loading chitosan improved viability that might be attributed to coating effects of chitosan and its low toxicity. In sum, viability was almost 100.00%  Wound area. Figure 9A,B shows the wound contraction (%) in different groups on days 3, 7, and 12. Data analysis did not show significant differences (P = 0.925) between the groups on day 3. The highest wound con-  LPS results. Figure   Pathology results. Table 2 shows the effects of Mlt, ZnO/Mlt, and ZnO/Mlt/Chsn ointments on pathology parameters. The treated mice had lower edema and immune cells than the mice in the Cnl group on all days, except for the mice treated with Mlt ointment. The administration of ointments decreased immune cells and edema on all days. The number of vessels and fibroblast cells was significantly higher in the mice treated with polysporin, ZnO/Mlt, ZnO/Mlt/Chsn, and polysporin ointments than in the control mice on days 3 and 7 (Fig. 9D). Collagen scores were significantly (P < 0.05) higher in the mice treated with polysporin and ZnO/Mlt/ Chn on day 12 (Fig. 11). All the mice had higher collagen than Ctrl mice on day 7 (P < 0.05). Epithelium did not have any thickness on day 3. The results indicated that the epithelium scores were significantly higher in the mice treated with polysporin and ZnO/Mlt, ZnO/Mlt/Chsn than in the control group on day 12. Malachite decreased edema and immune cells. In literature review, there was no paper investigating the effects of malachite on histological parameters. Notably, malachite shows weak antibacterial properties that decrease the edema and promote the proliferative phase. The results of histological parameters for ZnO are similar to the results reported by Ehsani et al. 24 . Anti-inflammatory properties and proliferative promoting effects are the mechanism suggested for the wound healing activity of ZnO nanoparticles 24 . Regarding the effects of chitosan as the wound healing structure, it must be stated that it causes to produce blood vessels, growth factors, and activating endothelial cells 15 . Chitosan and ZnO are antibacterial structures that accelerate wound healing, decrease the inflammation, and move the wound toward the proliferative phase. All the ointments increased collagen synthesis, thereby contracting the wound and closing it.  www.nature.com/scientificreports/ The results of immunofluorescence staining and gene expression. The results of immunofluorescence staining showed that expression intensities of TNF-α and bFGF were significantly (P < 0.05) higher and lower in the control groups than in the other groups, respectively (Fig. 12). The administration of Mlt ointments significantly decreased the expression of TNF-α and increased the expression of bFGF. Compared to antibiotics, the mice treated with ZnO/Mlt and ZnO/Mlt/Chn ointments had higher and lower expression for bFGF and TNF-α, respectively. Indeed, Fig. 13 presents the results of the gene expressions of IL-1β, IL-6, IL-10, and TGF-β. The administration of ointments based on malachite reduced the expression of IL-1β and IL-6 (P < 0.05) compared to the control group. The administration of ZnO/Mlt and ZnO/Mlt/Chn ointments significantly (P < 0.05) increased the expression of IL-10 on days 3 and 7, and TGF-β on day 7 compared to the control group. The lowest expression for IL-1β and the highest expression for IL-10 were observed in the mice treated with ZnO/Mlt/ Chsn ointment and polysporin. The increased expression of IL-1β in the wound prolongs the pro-inflammatory condition and hinders the wound healing process 29 . IL-6 is a pro-inflammatory cytokine and causes the inflammation. TNF-α, as a pleiotropic cytokine produced by keratinocytes, macrophages, and mast cells, delays the wound healing process 31 . IL-10 acts in contrast to TNF-α and accelerates the wound healing process by reducing the inflammation 32 . Additionally, bFGF stimulates the proliferative phase and the participation in synthesis of new vessels 33 . TGF-β promotes the proliferative phase and accelerates the wound healing process 34,35 . Briefly, the increased expression of IL-1β, IL-6 and TNF-α increases the inflammation and retards the wound healing process. Based on the findings, ointments produced from malachite significantly decreased the expression of IL-1β, IL-6 and TNF-α and increased the expression of IL-10, bFGF and TGF-β. In other words, ointments precede the wound healing process by molecular mechanisms that could be attributed to their compounds, while commercial antibiotics show their effects by antibacterial activity.  www.nature.com/scientificreports/

Conclusions
In sum, ZnO/Malachite nanocomposites were synthesized with the help of M. pulegium extract and its coated chitosan-derivatives to treat infected wounds. Physicochemical characterization of the prepared nanocomposites proved their successful synthesis. The prepared nanocomposite had antibacterial and antioxidant properties that accelerated the wound healing process and could compete with the standard ointment of polysporin. This study was conducted on infected wounds in mice, and the results cannot be used for other wounds in mice. Although, this study was conducted on mice, the potential results of the current study pave the way for clinical studies to investigate the effects of NCs on wounds and their uses in combination with other commercial ointments.

Materials and methods
Materials and methods. The chemical agents were of analytical grade. Zinc chloride, and medium molecular weight chitosan (190,000-310,000 Da., 167 75-85% deacetylated) were purchased from Merk and Sigma companies, respectively. Medicinal grade of malachite powder (free from hazardous elements) was prepared from Iranshid Co. Mashhad, Iran. To investigate morphological properties, EDX (MIRA3 FEG-SEM, Czech Republic) was used. Hydrodynamic diameter andsize distribution were evaluated, and zeta potential of the samples was investigated by DLS (Nanotrac Wave, Microtrac Co. USA). FTIR spectra (Shimadzu model FTIR 8101N spectrometer) was also used.
Preparation of M. pulegium extract. Leaves of M. pulegium were collected from (in accordance with institutional and national guidelines) Botanical Garden of Tabriz University, washed with distilled water, shaken for 24 h, and placed in an ultrasound bath under reduced pressure. It was then exposed to microwave radiation and filtered to obtain the supernatant and stored in a refrigerator for further use based on other studies [13][14][15] .

Biofabrication of ZnO/Mlt-NC, and ZnO/Mlt/Chsn-NC and preparation of ointments.
To prepare ZnO/Mlt-NC, 0.20 g of ZnCl 2 was mixed with 0.60 g of malachite powder in 75 ml of the extract, and then vigorously stirred at 65 °C for 12 h. The obtained raw product was then separated after centrifuging. This precipitate was washed with distilled water and ethyl alcohol to remove the remained ions and other impurities. Afterward, it was dried at 70 °C for 1 h to prepare ZnO/Mlt/Chsn-NC. One gram of ZnO/Mlt-NC powder was gradually added into 50 mL protonated chitosan solution (acetic acid 1% w/w) at room temperature, stirred for 24 h, and ZnO/Mlt/Chsn-NC was obtained. The therapeutic ointments (2% w/w) were formulated as reported by Mahmoudabadi et al. 14 . In sum, a certain amount of NCs (2 g) was mixed with 98 g base ointment to prepare NC ointments.
In-vitro antibacterial assessments. Minimum  www.nature.com/scientificreports/ In this formula, OD test is the optical density of cells exposed to tested foils, and ODcontrol is the optical. To evaluate reactive oxygen species (ROS), the cellular reactive oxygen species detection assay Kit (Abcam, Cambridge, UK) was used, and all the protocols were conducted based on the producer Company. All the conditions for culturing and incubation were similar to those for LDH. Following the incubation, the samples were transferred to microcentrifuge tubes and centrifuged at 1200 rpm for 5 min. Then, 100 µL of the supernatants were transferred into 96-well plates, and 100 μL of the diluted DCFDA were added to each well and incubated for an additional 45 min at 37 °C in the dark. Production of DCF was evaluated by fluorescence spectroscopy with an excitation wavelength at 485 nm and an emission wavelength at 535 nm on an ELISA reader.
2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. 2,2-diphenyl-1-picrylhydrazyl analysis was used to investigate antioxidant activity as reported by Gharehpapagh et al. 13  In vivo infected wound activity evaluation. Experimental animals. The healthy BALB/c male mice (n = 90) aged 10-12 weeks with a weight of 28 ± 4 g were prepared. The animals had unlimited access to water and food. They were kept under lab conditions prior to the start of study as the conditioning period. The mice were kept under a lighting diet of 12 h. This study lasted for 12 days in accordance with the Iranian ethical guidelines for the use of animals. All the used protocols, such as study design, sample size, randomization, outcome measures, data analysis, experimental procedures, and report of results, were in agreement with the ARRIVE guidelines,. The protocols were approved by the Committee on the Ethics of Animal Experiments of Veterinary Faculty and the Islamic Azad University Council on Animal Care, Tehran, Iran (IR.IAU.SRB.REC.1399.180), and were in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no.85-23, revised 1996). We declare that all methods were performed in accordance with the relevant guidelines and regulations.
The induction of infected wound. To induce wound, the general anesthesia was conducted by administrating the ketamine (50 mg/kg)/xylazine (10 mg/kg) cocktail intraperitoneally. After induction of anesthesia, the hair was shaved, and a circular full-thickness wound (with a diameter of 7 mm) was created by surgical cutaneous punch on the skin, and 0.5 McFarland Standard suspension in Muller-Hinton media (Merk Company-Germany) containing 10 7 bacterial cell of S. aureus. Furthermore, P. aeruginosa was immediately administrated into the wound site as reported previously 15,24,39 . The treatment was started 24 h after inoculation of bacteria. The mice were divided into 5 groups and daily treated with 0.5 g of ointments of polysporin® (500 unit/g bacitracin and 10,000 unit/g polymixin B), Malachite (Mlt), ZnO/Mlt, and ZnO/Mlt/Chsn. A group was regarded as control (Cnl) and was not treated. To prepare ointments, 2 g Mlt, ZnO/Mlt, and ZnO/Mlt/Chsn were mixed with base ointment (soft white paraffin, Parsin Shimi Company-Iran), and 2%-ointments were prepared. The administration of ointments lasted for 12 days. The wound area was investigated with the help of a transparent paper over the wound as reported previously 14,15,24,39 . In summary, the wound site was measured by placing a transparent paper over the wound and tracing it using a graph sheet and formula calculations.
Bacterial colony counts on wound surfaces. Bacterial colony counts were investigated as reported previously 14,15,24,39 with the help of sterile swab and cultured on plate count agar on days 3, 7 and 12, and the results were reported as CFU/g of granulation tissue. In short, 0.1 g of the sample was crushed, minced, and homogenized in a sterile mortar containing 10 mL of sterile saline and then diluted in tubes containing 9 mL of sterile saline. They were then cultured on plate count agar, incubated and the colonies were calculated.
Histopathological assessment. To investigate histopathological parameters, the samples were prepared from granulation tissue, along with 1-2 mm 2 healthy tissue, fixed in formalin, embedded in paraffin, and stained with hematoxylin and eosin and sirius red staining, and they were investigated by a light microscope as reported previously on days 3, 7, and 12 14,15,24,39 . Two blinded pathologists investigated the samples by a microscope and reported the results.
Investigation of lipoprotein polysaccharide (LPS). LPS was investigated as recommended by the producer company and using commercial kits (MyBioSourcere: Catalog No: MBS452438).